In modern molecular biology, it is an important task to identify which molecules and which configuration thereof composes each cell or organelle, which molecules are created, where the molecules are transported, and how the molecules are changed and divided in each organelle. Cellular motility and growth are central to normal physiological processes. When cell motility and growth are restricted, negative consequences such as tumor formation may result.
In particular, it is important to know three-dimensional distributions of polymers or elements constituting a cell and know changes of the three-dimensional distributions for understanding cell structure and function of each organelle. A representative method for identifying the three-dimensional distribution of the polymer or elements constituting the cell is a fluorescence imaging technique.
According to the fluorescence imaging technique, after a target molecule in cells is attached with a fluorescent marker or the molecule is treated to express fluorescence itself by gene manipulation, a fluorescence image allows the three-dimensional distribution of the target molecule to be identified. A confocal laser scanning microscope is a device to which the fluorescence imaging technique is applied. However, when conducting the fluorescence imaging technique, a foreign substance such as a fluorescent gene or a fluorescent marker is required to be introduced into the cell to perform imaging in an artificial state rather than a natural state of the cell itself. In addition, in order to detect a specific molecule by fluorescence imaging, the specific molecule is necessary to exist in a cell at a predetermined concentration or more. Thus, it is impossible to identify a three-dimensional distribution of a molecule having a low concentration of the specific molecule even when the molecule is attached with a marker or genetically manipulated. Further, since a fluorescent image is to be seen, additional steps and analysis are necessary to confirm whether the target molecule is attached to the marker.
As a cell imaging method other than an optical method and a biochemical method, there is a method of studying the structure and constituent molecules of cells and organelles by using a scanning electron microscope (SEM), a transmission electron microscope (TEM), an atomic force microscope (AFM), a scanning tunneling microscope (STM), or the like.
In the case of using an SEM, secondary electrons or back scattered electrons having the highest probability of occurrence among various signals generated in a sample are detected when an electron beam is scanned over a sample surface such that a target specimen is observed. However, it is difficult to image molecules, and it is required that the specimen is frozen and coated with a metal having a high atomic number.
A TEM uses a deflected electron beam and the electron beam which has penetrated a specimen is magnified by an electron lens for observation. However, although molecules are imaged, imaging and analysis take a long time and it is required that the specimen is frozen and thinly sliced.
An AFM is a two-dimensional scanning microscope with a pyramid-shaped probe in contact with a surface of a specimen. In case of using an atomic power microscopy, atomic level imaging is possible for inorganic specimen and metallic specimen. However, in case of cells in which water accounts for 75 to 80% and cells in which growth medium exists externally thereof, a resolving power of the microscopy deteriorates rapidly. In addition, though an AFM has good resolving power, an imaging area thereof is very narrow, and in living cells, a shape of the cells changes spontaneously or under the influence of the probe of the microscope whereby the AFM may have low image reliability. In addition, in order to obtain atomic resolution, it is required that a molecule to be imaged is placed on a flat solid substrate and low-temperature, low-pressure, and vacuum atmosphere is required. Furthermore, with probes used in current AFMs, it is impossible to image molecules large in size and in height, like intracellular polymers.
An STM is a type of scanning probe microscope, which analyzes the shape of the surface of the specimen using tunneling current. An STM is capable of imaging at the atomic level, but it is required to be in vacuum state to operate and required to maintain conductivity whereby imaging is impossible for cells containing a large amount of water.
Accordingly, the inventors of the present invention have developed an apparatus and a method of generating a three-dimensional image of a polymer, in which a process of sequentially removing surfaces of polymers is unnecessary or minimized while making practical imaging of intracellular polymers possible.